This document discusses downstream processing (DSP) which encompasses the processes following fermentation that isolate, purify, and formulate the desired product for end use. DSP aims to maximize recovery yield, minimize costs, and efficiently and safely recover products to required specifications. It can be divided into distinct unit processes like filtration, centrifugation, extraction, chromatography, and drying that are linked to achieve product purification. The document describes various unit operations used in DSP and factors to consider when selecting recovery processes.

1. Down-stream processing
(DSP)
Presented by
Bilqees Fatima Ph.D.
Presented to
Dr. Asad-u-Rehman
INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY
G.C. UNIVERSITY Lahore
PAKISTAN
Bioprocess Techniques

2. BIOPROCESSES
 Bioprocesses make use of living cells and microorganisms as well as
enzymes for the production of variety of bulk organic chemicals, food
products, pharmaceuticals and fuels.
Down stream processing
Up stream processing

3.  Down stream processing encompasses all processes
following the fermentation by which the desired product is
Isolated, Purified and Formulated for different end uses.
 Maximum recovery yield
 Minimum costs
 Efficient reproducibility
 Safely recovering the target product to the required
specification
Down Stream Process (DSP)
Objectives

4. Foam separation
Filtration
Centrifugation
Cell disruption
Fractional
Precipitation
Dialysis
Ultrafiltration
Electrophoresis
Distillation
Reverse osmosis
Diafiltration
Precipitation
Gel Filtration
Aqueous two phase
extraction
Sterile filtration
Crystallization
Lyophilization
Drying Packaging
Stages of Down Stream
Process
Downstream processing can be divided into a series of
distinct unit processes linked together to achieve
product purification

5. The choice of recovery
process
 The intracellular or extra-cellular location of the product
 The concentration of the product in the fermentation broth
 The physical and chemical properties of the desired product (to
selecting separation procedures)
 The intended use of the product
 The minimal acceptable standard of the product or broth
 The impurities in the fermented broth
 Speed of operation may be the overriding factor because of the
labile nature of some bioproducts
 Correct type and size of processing equipment
 The marketable price for the product
One is trying to obtain a high quality product as quickly as possible at
an efficient recovery rate using minimum plant investment
operated at minimum costs.

6. Separation mechanisms available at
different stages of DSP
SEAPARATION FACTOR BIOPROCESSING TECHNIQUES
Particle size
Molecular size and weight
Density differences
Temperature
Diffusion
Solubility
Ionic charge
Hydrophobicity
Electrophoretic mobility
Isoelectric point
Free thiol groups
Biospecific and biomimetic
interactions
Filtration, screening, microfiltration
Ultrafiltration, gel filtration
Ultracentrifugation, centrifugation, cyclone separation,
sedimentation
Precipitation by thermal denaturation
Reverse osmosis, dialysis
Solvent extraction, precipitation
Ion-exchange chromatography, electrophoresis
Precipitation, hydrophobic interaction chromatography,
reverse phased chromatography
Electrophoresis
Chromatofocusing, isoelectric focusing
Covalent chromatography
Affinity and pseudo-affinity chromatographic
techniques

7. Fermentation
Foam separation
Cell separation
Centrifugation, Filtration
Harvest cells Spend medium
Periplasmic product Extra-cellular product
Cell disruption Concentration steps
Centrifugation, Ultrafiltration
Cell extract Inclusion bodiese Medium concentrate
Product purification
Dialysis, Precipitation, Partition Chromatographic steps, Ultrafiltration,
Distillation, etc.
Finishing processes
Crystallization, Drying, Lyophilization, Sterile filtration, Packaging, etc.
Finished product
Primary recovery
Down stream processing

8. Foam Separation
 Depends upon differences in surface activity materials
 It may be possible to make some materials surface active:
 by the application of surfactants such as
 long chain fatty acids
 amines and
 quaternary ammonium compounds
 Colligends
 Collectors
 The important variables, which may need experimental
investigation, are:
 pH
 Air-flow rate
 Surfactants and
 colligends-collector ratio

9. Precipitation
 Precipitation involves the conversion of the soluble solutes in to
insoluble solids.
 subsequently separated from the liquid by
 filtration
 centrifugation
 Typical agents used in precipitation render the compound of
interest insoluble, these include:
 Acids and bases
 Salts (such as ammonium and sodium sulphate)
 Organic solvents (methanol, Chilled ethanol and acetone )
 Non-ionic polymer such as polyethylene glycol (PEG)
 Polyelectrolytes (triazine dye)
 Affinity precipitatants are an area of much current interest

10. Filtration (solid liquid separation)
 Filtration is a processes to separate suspended particles from liquid or
gas, using a porous medium, which retains the particles but allows
the liquids or gas to pass through.
 Concentration of particulates = 0.1-60%
 Size of particles = 1μm-1mm
 Coagulation (acid/base, change pH, charged particles)
 Flocculation (FeCl3/Al2(SO4)3, Van der Waals & London forces)
Filter aids: wood pulp, starch
powder, cellulose, inactive carbon,
diatomaceous earth and perlites.
Concentration = 0.5-5% (w/w)
Particle size = 2-20μm
Filter media
Rigid media: sand, gravel diatomaceous
earth, chacoal.
Porous media: carbon, porcelain, alumina,
metal sheets, wire mesh.
Flexible media: woven fabrics

11. Plate and frame filters
 The plates are covered with
filter media made up of cloth
or membrane
 Widely used as a polishing
device in breweries.

12. Pressure leaf filters
 Each metal leaf is consists
of a metal frame work of
grooved plates covered
with fine wire mesh or
filter cloth often precoated
with cellulose fiber.
 Suitable for fermented
broth with low solids
content and low
resistance to filtration

13. •Drum covered with fabric or metal filter mesh
•Drum revolve slowly at about 1 rpm
•Drum filter may be coated with a thick layer of diatomaceous earth or an inert
microporous material
•Used involving large volume of feed

14. Rotary vacuum filters
Fungal mycelia
produce fibrous
filter cake which
can easily be
separated with
string discharge
Yeast cells can
be collected
with scraper
discharge
The filter cloth can
be blocked by
bacterial cells or
mycelia. This
problem can be
overcome by
precoating
Mechanism of cake discharge

15. Liquid-liquid Extraction
(Difference in densities)
 This method is used while
separating two or more
immiscible liquids with
different densities.
 Mixture is taken in a
separating funnel.
 Allowed the mixture to stand
for some time.
 This separates the liquids
into layers.

16. Liquid-liquid Extraction
(Difference in solubility)
 Here work a simple rule “like
dissolve like”
 Polar liquids mix with each
other and dissolve salts and
other polar solids.
 The solvents for non-polar
compounds are liquids of low
or nil polarity.
 Dielectric constant is the
measure of degree of molar
polarization of a compound.
Contied…..

17. Liquid-liquid Extraction
(Difference in solubility)
 The final choice of solvent will be
influenced by the distribution or
partition coefficient K where
 K = Concentration of solute in extract
 Concentration of solute in raffinate
 K defines the ease of extraction.
 high value of K,
 good stability of product
 good separation of the aqueous
and solvent phase
 single stage extraction system
Solvents Dielectric constants
Hexane
Cyclohexane
Carbon
tetrachloride
Benzene
Diethyle ether
Chlorofprm
Ethyl acetate
Butane-2-ol
Butane-1-ol
Propane-1-ol
Acetone
Ethanol
Methanol
Water
1.90 (least polar)
2.02
2.24
2.28
4.34
4.87
6.02
15.8
17.8
20.1
20.7
24.3
32.6
78.5 (most polar)
However, the use of organic
solvents has limited application in
the bioprocessing of sensitive
bioproduct.

18. Aqueous Two-phase
Separation
 Phase separation occurs
when hydrophilic polymers
are added to an aqueous
solution, and concentration
exceed a certain value, two
immiscible aqueous phases
are formed.
 Have high water contents
 Low interfacial surface
tension
 Regarded as being
biocompatible
System Example
Non-ionic polymer
/non-ionic
polymer /water
Polyethylene glycol
/ Dextran
Polyelectrolyte
/non-ionic
polymer /water
Sodium carboxymethyle
cellulose
/polyethylene glycol
Polyelectrolyte
/Polyelectrolyte/
water
Sodium Dextran sulphate
/sodium carboxymethyl
cellulose
Polymer
/low molecular
weight
component/water
Dextran
/propyl alcohol
A large variety of natural and
synthetic hydrophilic polymers are
used today

19. Distillation
 This method is adopted to
obtain both the solute and the
solvent from a solution.
 solute is a soluble solid.
 both evaporation as well as
condensation.
 The distillation flask is
attached to a Leibig’s
condenser
 when a little is left, it is heated
separately in an open
container leaving it to
evaporate completely.

20. Fractional Distillation
 This method is adopted to
separate two or more miscible
liquids whose boiling points are
quite different.
 The liquid with the lower boiling
point is heated first so that it
evaporates and after the vapors
pass from through the
condenser, they fall into a
container as liquid.
NOTE: the temperature remains
constant for each boiling liquid
till it evaporates completely more
or less.

21. Distillation
Vacume Distillation

22. Membrane Processes
 Membrane separation consists of different processes
operating on variety of physical principles and
applicable to a wide range of separations of miscible
components.
 Separation affected by the use of thin, selective, semi-
permeable barriers
 Ceramic and metal filters also perform similar task
Dialysis Microfiltration Ultrafiltration Nanofiltration
Reverse osmosis
Membrane Processes

23. Dialysis
 Dialysis is a diffusive process
employing a second liquid
(dialysate) on the opposite side
of the sample.
 The rate of transport of a salt
through dialysis membrane
diminishes as salt concentration
in sample declines during the
process.
•Dialysis membrane is usually made of
cellulose acetate
•Pore size = 1-20 nm in diameter

24. Ultrafiltration
 Ultrafiltration is a selective
fractionation process utilizing
pressures up to 145 psi (10 bar).
 It concentrates suspended solids
and solutes of molecular weight
greater than 1,000.
 UF is widely used in
 fractionation of milk and whey,
 protein fractionation.
Fig. Cross-section of
anisotropic UF membrane.

25. Microfiltration
 Microfiltration is a low-pressure (10-
100psig) cross-flow membrane process
for separating colloidal and suspended
particles in the range of 0.05-10 microns.
 Crossflow microfiltration is normally
considered when the suspension to be
filtered proves difficult to process in a
centrifuge.
 Economically viable for bacterial cells (as
compared to centrifugation) and
filamentous cells (as compared to pre-
coat rotary vacuum filtration).
 shear sensitive animal cells,
plasmapheresis, sterile filtration of
pharmaceuticals.
Fig. Cross-section of
microporous membrane.
Particles are trapped on its
surface or within pores.

26. Applications of micro-filtration
Application Permeate Concentrate(Retentate) Benefits of MF
Cranberry juice clarification Low turbidity, clear
juice
Juice, suspended solids,
colloidal haze particles.
Removes suspended
solids and turbidity while
allowing the passage of
color, sugar and taste.
Wine filtration Low turbidity, clear,
flavorful wine
Wine, suspended solids,
colloidal haze particles.
Removes suspended
solids and turbidity while
allowing the passage of
color, alcohol and taste.
Industrial Waste Water
Treatment
Water & dissolved
solids
Water, suspended solids,
insoluble metal hydroxide
solids.
Removes suspended
solids and insoluble
metal hydroxide solids
from wastewater to allow
the permeate to be
discharged to a local
POTW.
Fermentation broth
clarification
Water & dissolved
solids
Water & suspended solids Removes suspended
solids from the
fermentation broth
leaving clarified liquid.

27.  Reverse osmosis is a high-
pressure, energy-efficient technique
for dewatering process streams,
concentrating low-molecular-weight
substances in solution, or purifying
wastewater.
Pressure requirement
•2–17 bar (30–250 psi)
for fresh and brackish
water
•40–70 bar (600–1000
psi) for seawater

28. Reverse Osmosis Membrane
• The most commonly
used is a spiral
wound
• CTA (cellulose tri-
acetate), which is
chlorine tolerant,
• TFC/TFM (thin film
composite/material),
which is not chlorine
tolerant. Reverse Osmosis Membrane

29. Applications of reverse
osmosis
 Desalination of
seawater
 Drinking water
purification
 wastewater purification
 Food Industry
 Car Washing
 Maple Syrup Production
 Hydrogen production
 Reef aquariums
approximately 54-42% of the water to
be removed from the sap
to prevent formation of minerals on the
surface of electrodes
for artificial mixture of seawater
to desalinate seawater or brackish
water to obtain drinking water
to prevent water spotting
6% total solids to 10–20% total solids
effluent in larger volumes (more than
42 cu. meter per hr)
chemical, biological, radiological, and
nuclear

30. Nanofiltration
 Nanofiltration is a special process selected when
RO and UF are not the ideal choice for separation.
 NF can perform separation applications that are
not otherwise economically feasible, such as
demineralization, color removal, and desalination.
 In concentration of organic solutes, suspended
solids, and polyvalent ions.
 the permeate contains monovalent ions and low-
molecular-weight organic solutions like alcohol.

31. MEMBRANE PROCESSES

32. Crystallization
 Crystallization is a process where solid particles of of specified size
and shape are formed from a homogeneous phase.
 Crystallization process consists of two steps
 Nucleation
 Crystal growth
 Super saturated state consists of three zones
 Metastable zone
 Intermediate zone
 Labile zone
 Crystallization may be initiated by
 Homogenous nucleation
 Hetrogenous nucleation
Dust, gas bubbles,
mechanical shock or
ultrsonic shock may also
bring about hetrogenous
nucleation
Crystal growth will occur subsequent
to nucleation or addition of seed
material.

33. Drying
 Drying removes the solvent,
mainly water, from the desired
product and in most cases
stabilizes the product making it
amenable for storing, packaging
or formulation.
 Drying takes place by movement
of water vapour from the
saturated surface through to the
stagnant air film in to the main
stream of drying air.
 The rate of drying depends on
rate of heat transfer to the drying
surface.
E
D
C B
A
A
Free moisture
Drying
rate
0

34. Drum Drier
 contact drier in which product is
contacted with a heated surface
 used for more temperature stable
products
 solid is in contact with the heating
surface for 6-15 seconds
 heat transfer coefficients are
generally between 1-2 kWm-2 K-1

35. Spray Drying
 Is most widely used for
drying of biological materials
when the starting material is
in the form of a liquid or
paste.
 The droplets then fall into a
spiral stream of hot gas at
150-250°C.
 The high surface area:
volume ratio of the droplets
results in a rapid rate of
evaporation and complete
drying in a few seconds.